【摘要】 目的:模拟结核分枝杆菌体内缺氧和微营养的生长环境建立其休眠模型。方法:将牛分枝杆菌卡介苗野生株BCG菌株在"微需氧+pH5.0+10%Dubos"的条件下培养,以菌株生长停滞,菌体表面脂质体增厚为休眠标准进行鉴定。取不同时间菌株测定600 nm下的吸光度(A)值绘制生长曲线,同时进行金胺O-尼罗红(Auramin-O,Nile Red)双荧光染色和实时荧光定量PCR(Quantitative Realtime-PCR,qRT-PCR)检测休眠相关基因的表达量,确定菌株进入休眠状态。结果:在休眠条件培养过程中,BCG菌株的生长速度与常规条件培养菌株相比明显减慢(P<0.01),并且在休眠条件培养中第10、14和18天细菌生长基本接近停滞状态(P<0.01);同时金胺O荧光染色显示绿色荧光减弱,尼罗红红色荧光增强;休眠相关基因hspX、devR表达量逐渐上调,且培养第18天菌株明显高于第0天培养菌株(P<0.05)。结论:应用"微需氧+p H5.0+10%Dubos"条件培养,以金胺O-尼罗红染色和devR表达为标准,可以成功建立结核分枝杆菌休眠模型。更多还原

【Abstract】 Objective: To build the in vitro multiple-stress dormancy model for mycobacterium tuberculosis based on the stresses within the host.Methods: Applied M.bovis BCG with combined stresses of low oxygen（5%）,high CO₂（10%）,low nutrient（10% Dubos medium） and acidic pH（5.0） to incubate and harvest at diffenert time point（0 to 18 days）,then to carry out the growth curve by detect the OD600 at different time point.Dual staining of Mtb with the combination of Auramine-O and Nile Red has been used to examine acid-fastness of Mtb cells and lipid body accumulation within the Mtb cells.The expression of dormancy related genes were checked by quantitative RT-PCR.Results: Comparing to conventional strains,the growth of dormancy strains was significantly decelerated at all phase（P<0.01）.The only acid-fast stain（green） positive cells gradually decreased and the other type steadily increased during multiple-stress treatment.The expression of dormancy related genes,hsp X and dev R was up-regulated at 0 to18 days,especially the expression of 18 day was significantly higher than the start day（P<0.05）.Conclusions: Mycobacterium tuberculosis multiple-stress dormancy model was successfully built.更多还原